Differentiation‐dependent expression of CA V and the role of carbonic anhydrase isozymes in pyruvate carboxylation in adipocytes

Abstract

The incorporation of radioactivity from 14C-labeled compounds into metabolic intermediates and total lipids was examined in 3T3 adipocytes. The heterocyclic sulfonamide carbonic anhydrase inhibitor (SCAI) 6-ethoxyzolamide (ETZ) caused a decrease (42+/-7% of control, IC50 = 2.2+/-1.1 x 10(-7) M) in the incorporation of [14C] bicarbonate into several Krebs cycle intermediates in 3T3-F442A adipocytes. This decrease in pyruvate carboxylase-mediated [14C] carbon fixation was associated with a reduction in fluorometrically determined [citrate] and [malate]. The ability of ETZ to decrease both the incorporation of radioactivity into and the concentrations of Krebs cycle intermediates was not of sufficient magnitude to lower [ATP], but was associated with a decrease in de novo lipogenesis from [14C]glucose. De novo lipogenesis was also inhibited to a similar extent by trifluormethanesulfonamide, an aliphatic SCAI, which suggests that the effects are mediated by carbonic anhydrase. ETZ did not inhibit de novo lipogenesis from [14C]glutamine (12.38+/-1.068 nmol/mg protein, ETZ; 12.5+/-0.846 nmol/mg protein, DMSO). This suggests that ETZ inhibition of lipogenesis involves an inhibitory effect on pyruvate carboxylase as opposed to acetyl CoA carboxylase, because the incorporation of glutamine into lipids does not involve pyruvate carboxylase. Decreased de novo lipogenesis was also observed by incubating cultures in media that contained 1 mM bicarbonate (atmosphere:100% humidified air) rather than 25 mM bicarbonate (atmosphere: 95% humidified air/5% CO2). This suggests that exogenous CO2/bicarbonate may be required to sustain maximal rates of de novo lipogenesis. Because these results implied that CA V, the mitochondrial isoform of carbonic anhydrase, might be present in adipocytes, CA V levels were measured by immunoblotting. Mitochondrial preparations of adipocytes and liver were found to contain similar concentrations of CA V. Unlike adipocyte CA III, CA V concentrations were not significantly different in lean and obese Zucker rats. However, CA V levels were ninefold higher in differentiated 3T3-F442A adipocytes compared to undifferentiated adipoblasts. Our data indicate that CA V is relatively abundant in adipocyte mitochondria and exhibits differentiation-dependent expression like pyruvate carboxylase and the cytosolic isozymes CA II and CA III. The possible roles of CA II and CA V in pyruvate carboxylation are discussed.

Formation of chlorinated lipids post-chlorine gas exposure

“…Changes in lung endothelial and/or epithelial permeability were indicated by increased BAL protein levels, consistent with the pro-permeability effects of Cl-lipids. Also, similar to Cl 2 gas, Cl-lipids increased levels of inflammatory cells; however, these were not HOCl reactions (44,45), it is likely that both Cl 2 and Cl 2 -derived HOCl mediate formation of Cl-lipids during Cl 2 gas exposure. A significant role for MPOderived HOCl can be excluded, however, based on similar Cl-lipid levels in neutropenic mice exposed to Cl 2 gas.…”

Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism

“…Pharmacological studies with CA inhibitors have implicated mitochondrial CAs in several metabolic pathways, including gluconeogenesis, ureagenesis, and lipogenesis (14)(15)(16)(17). The common thread is the requirement in mitochondria for HCO 3 − , which does not freely cross the inner mitochondrial membrane (29), in contrast to CO 2 , which is freely permeable.…”

“…Price et al (34) used these mice to show the importance of mitochondrial CAs in producing the metabolites that lead to oxidative stress. Also, CA inhibitors have been shown to inhibit lipogenesis in hepatocytes (35) and adipocytes (17), and the effects are suggested to be due to inhibition of CA V. The presence of other CA isoforms in these tissues, notably CA II and CA III among others, has made it difficult to pinpoint the critical CA targeted by the inhibitors. Studies in tissues from the CA V-deficient mice reported here could help resolve this question.…”

“…13 and 14). Inhibition of both processes by CA inhibitors provided pharmacological evidence that mitochondria require a CA for these metabolic processes (15)(16)(17)(18)(19).…”

QSARs on human carbonic anhydrase VA and VB inhibitors of some new not yet synthesized, substituted aromatic/heterocyclic sulphonamides as anti-obesity agent

“…These two isozymes are involved in several biosynthetic processes, such as ureagenesis, gluconeogenesis and lipogenesis 1,2,[6][7][8] . As hCA VA/VB are involved in several biosynthetic processes catalysed by pyruvate carboxylase, acetyl CoA carboxylase and carbamoyl phosphate synthetases I and II, providing the bicarbonate substrate to these carboxylating enzymes involved in fatty acid biosynthesis, their inhibition may lead to the development of anti-obesity agents possessing a new mechanism of action 8 .…”